The local ethics committee at the University Medical Center approved this study (approval number: BB166/17). Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy donors provided by the Institute of Transfusion Medicine Greifswald by density-gradient centrifugation using lymphocyte separation medium (VWR, Germany). Residual erythrocytes were lysed using a red blood cell (RBC) lysis buffer (Thermo Fisher, Germany). For monocyte isolation, CD14 microbeads (Miltenyi Biotec, Germany) were used according to the manufacturer's protocol. Flow cytometric (Attune NxT; Applied Biosystems, USA) verification of purity was performed immediately after isolation and was always greater than 85%. 1 × 105 monocytes per well were seeded in 24-well plates (Sarstedt, Germany). The cell culture medium was supplemented with 800 IU granulocyte–macrophage colony-stimulating factor (GM-CSF) and 500 IU interleukin (IL) 4 (all PeproTech, Germany) to initiate monocyte-derived dendritic cells (DCs). After two days, cells were re-stimulated by adding the same amounts of GM-CSF and IL-4 to the cell culture medium. Immature DCs were used for co-culture experiments on day 5.
Interleukin il 4
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom
Interleukin (IL)-4 is a cytokine that plays a crucial role in the immune system. It is a soluble protein secreted by various cell types, including T cells, mast cells, and basophils. The primary function of IL-4 is to promote the differentiation of naive T cells into Th2 cells, which are involved in the humoral immune response and the production of antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Gm csf, by Thermo Fisher Scientific (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Granulocyte macrophage colony stimulating factor gm csf, by Thermo Fisher Scientific (5 mentions)
Ifn γ, by Thermo Fisher Scientific (5 mentions)
Il 13, by Thermo Fisher Scientific (5 mentions)
Interferon ifn γ, by Thermo Fisher Scientific (5 mentions)
Cd14 microbead, by Miltenyi Biotec (4 mentions)
Lipopolysaccharides (lps), by Thermo Fisher Scientific (4 mentions)
Lipopolysaccharides (lps), by Merck Group (4 mentions)
Lipopolysaccharide lps, by Merck Group (3 mentions)
Attune nxt, by Thermo Fisher Scientific (2 mentions)
Lymphocyte separation medium, by Avantor (2 mentions)
24 well plate, by Sarstedt (2 mentions)
Granulocyte macrophage colony stimulating factor, by Thermo Fisher Scientific (2 mentions)
Histopaque 1077, by Merck Group (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Il 1β, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
33 protocols using interleukin il 4
1
Generation of Monocyte-Derived Dendritic Cells
2
Cell culture and macrophage polarization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
U87 human glioma cell line stable expressed red fluorescence protein (RFP) and luciferase (U87-RFP-Luc) was purchased from the Keyuandi, Biological Technology Development Co., Ltd. (Shanghai, China). MFC mouse forestomach cancer cell line labeled with green fluorescence protein (MFC-GFP), and mouse macrophage cell lines, RAW264.7 and Ana-1, were purchased from the Institute of Medical Sciences, Peking Union Medical College (Beijing, China). The cells were cultured in RPMI 1640 medium and Dulbecco’s modified Eagle’s medium (DMEM), respectively, supplemented with 10% fetal bovine serum (FBS) (Gibco, ThermoFisher Scientific, USA) and 100 units/ml penicillin, and 100 units/ml streptomycin in a humidified atmosphere of 5% CO2 at 37 °C. All protocols using human cell lines were approved by the Research Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, and conducted in accordance with the approved guidelines for safety requirements. M1 macrophages were polarized by stimulating with 10 ng/ml lipopolysaccharide (LPS) (Sigma-Aldrich) and 20 ng/ml interferon (IFN)-γ (Peprotech). M2 macrophages were polarized by stimulating with 20 ng/ml interleukin (IL)-4 (Peprotech).
3
Generating Gr-1+CD11b+ Cells from Bone Marrow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Induction of Gr-1+CD11b+ cells from bone marrow progenitors was done as described previously,21 (link),22 with mild modifications. In brief, bone marrow cells were obtained from the femurs and tibias. C-kit+lineage− progenitors were sorted by FACS and cultured in 24-well plates in medium conditioned by the supernatants from colon explants or recombinant murine GM-CSF (50 ng/mL; Peprotech), supplemented with 10% fetal calf serum (Gibco) and interleukin (IL)-4 (20 ng/mL; Peprotech). In some experiments, purified neutralizing anti-GM-CSF monoclonal antibody (1 µg/mL; Biolegend) was added into the culture. Cells were collected 7 days later, and the proportion of Gr-1+CD11b+ cells was analyzed by flow cytometry.
4
Autologous Dendritic Cell Vaccine for HPV18 E7 Peptide Immunotherapy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Autologous dendritic cells (DCs) were generated as previously described, with minor modifications [25 (link),26 (link)]. PBMCs were incubated for 2 h at 37°C using complete RPMI medium containing 10% fetal bovine serum (FBS). Adherent monocytes were resuspended at a concentration of 5 × 106 cells/mL in complete RPMI medium with granulocyte-macrophage colony-stimulating factor (1500 IU/mL; PeproTech, Rocky Hill, NJ, USA) and interleukin (IL)-4 (1200 IU/mL; PeproTech). On days 2, 4, and 6 of culture, fresh cytokines were added. On day 5 of culture, 10 ng/mL of tumor necrosis factor-α (R&D Systems, Minneapolis, MN, USA) was added for DC maturation. After maturation, autologous DCs were pulsed with peptides for at least 6 h. PBMCs were plated at a concentration of 2 × 106 cells per well in a 24-well culture plate (Nunc, Rochester, NY, USA) with 2 mL of complete RPMI medium. PBMCs were sensitized with synthetic HPV18 E7 peptides (10 μg/mL/well), and 1000 IU/mL/well of recombinant human IL-2 (rhIL-2; PeproTech) was added. Additionally, rhIL-2 (1000 IU/mL/well) was added to the culture every other day. For 1-week expansion, peptide-pulsed autologous DCs (4–10 × 106/well) were added to the PBMCs on day 7, incubated for 6 h, and analyzed with flow cytometry. DC-treated PBMCs were cultured for another 7 days, and cytotoxicity assays were performed.
5
Murine GM-CSF and IL-4 Modulation of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant murine granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 were purchased from PeproTech (NJ, USA). Rat anti-mouse fluorescence-conjugated CD3, CD4, CD8, CD25, Foxp3, interferon (IFN)-γ, IL-17, CD11c, CD40, CD80, CD86, CD83, and MHC-II (I-A/I-E), as well as their corresponding isotype controls, were purchased from BD PharmingenTM (San Diego, CA, USA). Microbead-conjugated anti-CD11c and LS separation columns were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Anti-mouse insulin and glucagon monoclonal antibodies (mAbs) were purchased from Cell Signaling Technology (Danvers, US). The lentiviral short hairpin RNA vector (shRNA) Dectin-1-RNAi-GFP was constructed by Shanghai Genechem Co., Ltd. (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits for measurement of INF-γ, IL-10, and IL-17 were purchased from CUSABIO (Wuhan, China), and a kit for measuring tumor growth factor (TGF)-b was purchased from eBioscience. Streptozotocin (STZ), lipopolysaccharide (LPS), and Histopaque1077 were purchased from Sigma-Aldrich (St. Louis, CA, USA). Diphenylthiocarbazone (DTZ), acridine orange (AO), propidium iodide (PI), Liberase TL, a blood glucose meter, and blood glucose test strips were purchased from Roche (Basel, Switzerland).
6
Hematopoietic Stem Cell-Derived Dendritic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To generate hematopoietic stem cell-DCs (Stem-DCs), lineage-negative cells were selected from BM-MNCs by magnetic beads (MACS lineage depletion kit, Miltenyi Biotech, Germany) as HSCs. HSCs were seeded at a concentration of 1 × 106 cells per well in 24-well plates and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, granulocyte–macrophage colony-stimulating factor (GM-CSF, 100 ng/mL), stem cell factor (SCF, 50 ng/mL), and FMS-like tyrosine kinase 3 ligand (Flt3L, 50 ng/mL) (Peprotech, Rocky Hill, NJ, USA) for 7–10 days to promote their proliferation and differentiation into the monocyte lineage. Then, the cells were placed in culture media containing GM-CSF (1000 U/mL) and interleukin (IL)-4 (1000 U/mL) (Peprotech, USA) for 4–5 days to induce their differentiation into DCs.
7
Monocyte-Derived Macrophage Polarization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For differentiation of monocytes to macrophages and further polarization towards M1 and M2 phenotypes, human primary monocytes were first stimulated with 20 ng/mL GM-CSF or M-CSF (Peprotech, Hamburg, Germany) for 6 days in macrophage medium (RPMI 1640 supplemented with 10% FCS, 2 mmol/L l -glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin) to obtain monocyte-derived macrophages. These macrophages were further polarized for 48 h in macrophage medium with 100 ng/mL lipopolysaccharide (LPS) and 20 ng/mL interferon (IFN)-γ (Peprotech, Hamburg, Germany) to obtain M1 or with 20 ng/mL interleukin (IL)-4 (Peprotech) to obtain M2 phenotypes22 (link).
8
Generating Mature Dendritic Cells from PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMCs) of HLA-A*11:01-positive, healthy donors were isolated by density-gradient centrifugation from buffy coats (Guangzhou Blood Center) and cryopreserved. CD14+ cells were obtained by magnetic separation (BioLegend) from cryopreserved PBMCs to generate DCs.
The CD14+ cells were then cultured in complete RPMI-1640 medium containing 800 IU/mLgranulocyte macrophage colony stimulating factor (PeproTech) and 500 IU/mL interleukin (IL)-4 (PeproTech). The cultures were fed with fresh medium and cytokines every 2–3 days. Mature DCs were generated by adding 10 ng/mL IL-1β, 10 ng/mL IL-6, 10 ng/mL tumor necrosis factor alpha, and 1 µg/mL PGE2 for 48 hours from day 5.
The CD14+ cells were then cultured in complete RPMI-1640 medium containing 800 IU/mLgranulocyte macrophage colony stimulating factor (PeproTech) and 500 IU/mL interleukin (IL)-4 (PeproTech). The cultures were fed with fresh medium and cytokines every 2–3 days. Mature DCs were generated by adding 10 ng/mL IL-1β, 10 ng/mL IL-6, 10 ng/mL tumor necrosis factor alpha, and 1 µg/mL PGE2 for 48 hours from day 5.
9
Generating Bone Marrow-Derived Dendritic Cells
10
Bone Marrow-Derived Macrophage Polarization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bone marrow cells were obtained by flushing the femurs and tibias of normal male C57/BL6 mice in Dulbecco's modified Eagle's medium (DMEM; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 30% L929 conditioned medium at 37°C. The medium was changed at day 3 and day 5. L929 conditioned medium was the supernatant from growing L929 cells in DMED-containing 10% FBS for 5 days. After 7 days incubation, bone marrow-derived macrophages (BMDMs, M0) were harvested. 100 ng/ml lipopolysaccharide (LPS) and 6 ng/ml interferon (IFN)-γ were used to stimulate M0 and M1 macrophages which were harvested after 24 h. In addition, M2 macrophages were induced by stimulating M0 macrophages with 10 ng/ml interleukin (IL)-4 and 10 ng/ml IL-13 for 24 h. IL-4, IL-13, LPS and IFN-γ were purchased from PeproTech, Inc. (Rocky Hill, NJ, USA).
Pioglitazone and PPARγ antagonist GW9662 were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). M0 macrophages were stimulated with Pioglitazone (5 µm) for 24 h in the presence or absence of GW9662. PPARγ inhibitor GW9662 (5 µm) was added 6 h before treatment with Pioglitazone.
Pioglitazone and PPARγ antagonist GW9662 were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). M0 macrophages were stimulated with Pioglitazone (5 µm) for 24 h in the presence or absence of GW9662. PPARγ inhibitor GW9662 (5 µm) was added 6 h before treatment with Pioglitazone.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!